• Journal of Embryo Transfer
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• v.22 no.1
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• pp.1-7
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• 2007

This study was conducted to examine the role of intact cumulus cells during in vitro fertilization (IVF) on sperm penetration, male pronuclear (MPN) formation and subsequent embryo development of oocytes matured and fertilized in vitro. Cumulus-oocyte complexes obtained from the slaughtered gilt ovaries were matured for 44 h in TCM199 containing 10% porcine follicular fluid, epidermal growth factor and hormones. After maturation culture, denuded oocytes or oocytes with intact cumulus cells were coincubated with frozen-thawed boar semen for 8h in a modified tris-buffered medium containing 5mM caffeine and 10mM calcium chloride. Putative zygotes were fixed and examined for sperm penetration and MPN formation (Experiments $1{\sim}3$), or cultured in North Carolina State University-23 medium fo. 156 h (Experiment 3). In Experiment 1, sperm penetration was examined after insemination of denuded oocytes and oocytes with intact cumulus cells at the concentration of $7.5{\times}10^5$ sperm/ml. Optimal sperm concentration for IVF of cumulus-intact oocytes was determined in Experiment 2 by inseminating intact oocytes with $2{\sim}5{\times}10^6$ sperm/ml. In Experiment 3, denuded or intact oocytes were inseminated at the concentrations of $7.5{\times}10^5$ and $4.0{\times}10^6$ sperm/ml, respectively, and in vitro embryo development was compared. Sperm penetration was significantly (p<0.01) decreased in cumulus-intact oocytes compared to denuded oocytes (35.2% vs. 77.4%). Based on the rates of sperm penetration and normal fertilization, the concentration of $4.0{\times}10^6$ sperm/ml was optimal for the IVF of intact oocytes compared to other sperm concentrations. The presence of intact cumulus cells during IVF significantly (p<0.05) improved embryo cleavage (48.8% vs. 58.9%), blastocyst (BL) formation (11.0% vs. 22.8%) and embryo cell number $(22{\pm}2\;vs.\;29{\pm}2\;cells)$ compared to denuded oocytes. In conclusion, these results suggest that intact cumulus cells during IVF inhibit sperm penetration but improve embryo cleavage, BL formation and embryo cell number of porcine embryos produced in vitro.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.177-184
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• 2013

The objective of this study was to analyze the accuracy of estrus detection of heat detector and analysis of estrus behavior (mounting and mounted), and the evaluation of conditions required for improving reproductive efficiency in Holstein dairy cows fitted with a estrous detector. The heat detection system consists of estrous detector based on wireless sensor and an electric bulletin board displayed estrus behavior data. When cow mounting other cows, the accuracy of estrus behavior displayed an electric bulletin board were 87.5% (mounting other cows only), 100% (mounting other cows but not standing), 80.0% (mounting other cows with standing for 1~4 seconds), 90.0% (mounting other cows but not standing for 1~4 seconds), 80% (mounting other cows with standing for more than 5 seconds) and 90.0% (mounting other cows but not standing for more than 5 seconds). When cow mounted other cows, the accuracy of estrus behavior displayed an electric bulletin board were 100% (mounted other cows but not standing), 100% (mounted other cows with standing for 1~4 seconds), 100% (mounted other cows but not standing for 1~4 seconds) and 100% (mounted other cows with standing for more than 5 seconds). Circadian distribution of first observed in estrus were 59.1% (am 8~pm 6) and 40.9% (pm 6~am 8). Distribution for the number of estrus behavior were 40.9% (less than 3 times), 36.4% (4~6 times) and 22.7% (more than 4 times). The conception rates relative to interval from first estrus behavior to insemination for estrus periods were 23.1% (less than 11 hours) and 55.6% (12~20 hours).

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.5
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• pp.605-611
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• 2015

A synthetic strain of ducks (Anas platyrhynchos) was developed by introducing genes for long duration of fertility to be used as mother of mule ducklings and a seven-generation selection experiment was conducted to increase the number of fertile eggs after a single artificial insemination (AI) with pooled Muscovy semen. Reciprocal crossbreeding between Brown Tsaiya LRI-2 (with long duration of fertility) and Pekin L-201 (with white plumage mule ducklings) ducks produced the G0. Then G1 were intercrossed to produce G2 and so on for the following generations. Each female duck was inseminated 3 times, at 26, 29, and 32 weeks of age. The eggs were collected for 14 days from day 2 after AI. Individual data regarding the number of incubated eggs (Ie), the number of fertile eggs at candling at day 7 of incubation (F), the total number of dead embryos (M), the maximum duration of fertility (Dm) and the number of hatched mule ducklings (H) with plumage colour were recorded. The selection criterion was the breeding values of the best linear unbiased prediction animal model for F. The results show high percentage of exhibited heterosis in G2 for traits to improve (19.1% for F and 12.9% for H); F with a value of 5.92 (vs 3.74 in the Pekin L-201) was improved in the G2. Heritabilities were found to be low for Ie ($h^2=0.07{\pm}0.03$) and M ($h^2=0.07{\pm}0.01$), moderately low for Dm ($h^2=0.13{\pm}0.02$), of medium values for H ($h^2=0.20{\pm}0.03$) and F ($h^2=0.23{\pm}0.03$). High and favourable genetic correlations existed between F and Dm ($r_g=0.93$), between F and H ($r_g=0.97$) and between Dm and H ($r_g=0.90$). The selection experiment showed a positive trend for phenotypic values of F (6.38 fertile eggs in G10 of synthetic strain vs 5.59 eggs in G4, and 3.74 eggs in Pekin L-201), with correlated response for increasing H (5.73 ducklings in G10 vs 4.86 in G4, and 3.09 ducklings in Pekin L-201) and maximum duration of the fertile period without increasing the embryo mortality rate. The average predicted genetic response for F was 40% of genetic standard deviation per generation of selection. The mule ducklings' feather colour also was improved. It was concluded that this study provided results for a better understanding of the genetics of the duration of fertility traits in the common female duck bred for mule and that the selection of a synthetic strain was effective method of improvement.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.3
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• pp.199-205
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• 2020

Cryopreservation of semen is useful for animal breeding via artificial insemination (AI). However, the use of frozen-thawed boar semen is limited due to cryodamage. The aim of this study was to investigate the effects of different concentrations of MitoTEMPO (a mitochondria-targeted antioxidant) in lactose-egg yolk (LEY) extenders on kinetic characteristics of frozen-thawed boar sperms. Semen samples were collected from mature Duroc boars (2~3 years old) and cryopreserved in LEY extenders containing 0, 0.5, 5, 50, and 500 μM MitoTEMPO. The kinetic characteristics of frozen-thawed sperms were determined 0 and 30 min after thawing using computer-assisted sperm analysis (CASA). Results indicated that sperm motility immediately after thawing was significantly higher with 5 and 50 μM (50.46±2.71% and 46.96±2.66%, respectively) than with 500 μM MitoTEMPO (35.40±2.95%) (P<0.05). However, there were no significant differences in other kinetic characteristics except motility. In conclusion, the addition of MitoTEMPO to the sperm freezing extender may have a beneficial effect on motility of post-thawed boar semen.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.9-16
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• 2002

This study was carried out to investigate the effects of extenders such as Beltsville thawing solution(BTS), Modena and Androhep, preservation temperature and period of liquid boar semen on semen characteristics and reproductive performance. Boars were raised at Swine Artificial Insemination Center in National Livestock Research Institute, Sunghwan, Chungnam, Korea. This experiment was carried out from 1995 to 2000. The results obtained were summarized as follows. 1. Sperm motility in the samples with Androhep and BTS reduced from day 5 and in the samples with Modena reduced from day 3 of storage. pH or 3 extenders varied from 6.24 to 7.04 during day 1 to 5 of storage. Farrowing rate of sows inseminated with liquid boar semen offended with BTs, Modena and Androhep extenders did not show any differences until day f after semen collection. Sows inseminated with Androhep extender had better farrowing rates (P＜0.05) than those with Modena extender at day 1 or 5 after semen collection, but farrowing rates after AI using BTS did not differ compared to those Androhep and Modena. Litter size did not show any differences among the three extenders, but Androhep had the decreased litter size from day i of storage. 2. Motility and normal acrosome of the sperm preserved at 5$^{\circ}C$ did not show any differences until day 4 of storage, but those at 17$^{\circ}C$ changed from day 3 and 4, respectively. Farrowing rate of sows artificially inseminated with liquid boa. semen preserved at 17$^{\circ}C$ had higher, than at 5$^{\circ}C$ (p＜0.05), but there was no significant differences in litter size. Farrowing rates and litter size were decreased from day 2 and day 3 of storage at 17$^{\circ}C$, respectively. Farrowing rate of sows inseminated with the preserved semen at 5$^{\circ}C$ did not changed until day 4, but the litter size at 5$^{\circ}C$ was lower than that at 17$^{\circ}C$.

• Reproductive and Developmental Biology
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• v.32 no.1
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• pp.51-58
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• 2008

This study was performed to investigate the relation between birth weight and survivability on the production of cloned Hanwoo calves. The 580 cloned embryos were transferred into the 293 recipients. The pregnancy rate of the cloned embryos was 72.3% at 50 days after embryo transfer, and then the rate was dramatically decreased. The mean gestation lengths were 287 days in both clone (range of$279{\sim}295$ days) and artificial insemination (AI, range of $255{\sim}293$ days) calves, respectively. The mean birth weight of cloned calves (30.3kg) was significantly higher compared to that of AI calves (23.7kg) (p<0.05). Among the cloned calves, the birth weight was not different in both normal delivery (n=17, 29.9kg) and caesarean section (n=14, 32.3kg). The weight, however, was significantly higher in the clones (n=18, 32.8kg) dead within 175 days than that of the clones (n=11, 28.3kg) alive more than 175 days after birth (p<0.05). Interestingly, all cloned calves weighed <15kg (n=5) or >35kg (n=9) at birth have been dead within 175 days from the date of birth. The causes of death in the cloned calves were premature birth (n=2, 10.0%), abnormal function of lung and liver (n=2, 10.0%), abnormal function of lung (n=4, 20.0%), malformation (n=4, 20.0%), unknown (n=4, 20.0%), and sudden death syndrome (n=4, 20.0%), respectively. Our findings suggest that normal birth weight is one of the most important factors to survive more than 6 months in cloned calves.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.115-124
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• 2003

The objective of this study was to compare the effect of four different estrus induction methods in anestrus cows on the estrus induction and pregnancy following artificial insemination (AI). Sixty-five cows (3∼4 years old) were selected and divided into four different estrus induction treatment groups. Group 1, 12 cows were treated by Ovsynch program combined with GnRH and PGF$_2$a. Group 2, 12 cows were treated by "Tow plus Two" program with GnRH and PGF$_2$a. Group 3, 20 cows were treated by "Tow plus Two" program following intravaginal progesterone implantation (CIDR). Twenty one cows in Group 4 were treated by "Tow plus Two" program following follicular rupture and intravaginal progesterone implantation. Cows were then observed estrus induction and inseminated artificially at 12 h and 24 h after standing estrus. The rates of estrus induction in Group 4 (18/21, 86%) was significantly (P<0.05) higher than those in groups 1, 2 and 3 (8/12, 67%; 9/12, 75%; 14/20, 70%). In the mean time of onset of estrus after final administration of GnRH in different hormone-treated cows, the cows in Group 3 (24.2$\pm$2.2) and Group 4 (23.4$\pm$2.0) were significantly (P<0.05) shorter than that in Group 1 (28.5$\pm$4.6) and Group 2 (26.4$\pm$3.3). The rates of pregnancy diagnosed on Day 28 were significantly different between treatment groups. Significantly (P<0.05) higher rate of pregnancy was observed in Group 4 (17/20, 85.0%) than those in Groups 1, 2 and 3 (7/11, 63.6%; 8/12, 66.7%; 15/20, 75.0%, respectetively). The rate of abortion diagnosed on 49 days of gestation was significantly (P<0/05) lower in Group 4 (1/17, 5.9%) than those in Groups 1, 2 and 3 (2/7, 28.7%; 2/8, 25% and 3/15, 20%, respectively). In conclusion, combined treatments with GnRH and PGF$_2$a following follicular rupture and progesterone implant in anestrus cows was considered to be most effective in estrus induction and maintenance of pregnancy. Further studies are needed to verify the functional mechanisms of residual follicles in anestrus ovaries on retarding the response of hormonal treatments.sponse of hormonal treatments.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.139-143
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• 2007

This study was performed to investigate the reproductive characteristics of the cloned Hanwoo bulls produced by SCNT. The semen ejaculated from the cloned bulls (C-38 and C-39) and normal Hanwoo bull was properly measured the volume, the number of sperm, and the viability of frozen-thawed sperm. The sperm activity was analyzed using computer assisted sperm analysis (CASA). To analyze fertilizing ability of the cloned bulls, in vitro fertilization and artificial insemination were performed using the frozen-thawed semen. There were no differences in semen volume, sperm concentration, and the viability of frozen-thawed sperm between cloned bulls and normal bull. The difference was statistically significant in total motility, curvilinear velocity (VCL), straight-line velocity (VSL), and average-path velocity (VAP) of both cloned bulls compared to those of normal Hanwoo bull, respectively (p<0.05). The cleavage and blastocyst development rate were not different between the groups. five cloned cows were artificially inseminated using the frozen-thawed semen of C-38, two of them became pregnant. Two second generation calves (one male and one female) were produced. Based on these results, the cloned Hanwoo bulls showed normal reproductive abilities of semen parameters and sperm activity to their comparators and produced cloned calves, although there are some individual differences on the parameters.

• Development and Reproduction
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• v.5 no.1
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• pp.47-52
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• 2001

Predetermination of sex in livestock of offpring is in great demand and is of critical importance to providing for the most efficient production of the animal ariculture. Such a sexing techlology would also enhance the economy of conventional artificial insemination(AI) and aid the porcine industry. The purpose of this study was to evaluate the efficiency of enriching X-bearing porcine sperm using discontinuous percoll gradients and PCR mefhod. Semen was collected from mature boars of proven fertility center (AI center KimHae). Sperm was leaded on the isotonic discontinuous percoll gradient and then it was centrifuged at 120 ${\times}$ g for 20 minutes. After centrifugation, sperm included in each fraction were recovered (7${\times}$10$^6$ sperms/ml) and then sperm genomic DNA was extractedfor the PCR. SRY gene was used to evaluate the ratio between X and Y sperm in the separated fractions. Ju viro ffrtilization wascarried out by adding the unseparated sperm (control) or separated (experimental poop) to the matured oocytes in TCM-199. Embryos for sex determination were obtained at 2 cell stage and then was used for SRY gene amplification. After centrifugation of discontinuous percoll gradient, the most motile sperm was obtained at 95% fiaction (94.4% ${\pm}$ 5.1%, p < 0.01). The PCR analysis evaluated that 30%, 50% and 65% fractions were Y sperm rich, whereas 80% and 95% fractions were X sperm rich. PCR analysis with each porcineembryo showed that 33.3% of control and 66.7% of experimental group were determined as female embryos. In conclusion, in vitro matured oocytes inseminated with sperms (95% fraction) prepared by percoll gradient centrifugation showed high fertilization rates and female embryos than control sperms.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.169-176
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• 2005

Loop-mediated isothermal amplification (LAMP) is novel DNA amplification methods that amplifies a target sequence specifically under isothemal condition. The present study was to assess the in vitro viability afier biopsy and sexing rate of different types of embryo biopsied. In vivo compact morulae and blastocyst embryos were obtained from Korean Native Cow (KNC) superovulated with FSH (Antorin, R-10) on 7 Day after artificial insemination. in vitro compact morulae and blastocyst embryos were obtained with KNC or Holsteins that were gained on 6, 7 or 8 day after in vitro fertilization(IVF) with frozen semen. Biopsy of bovine embryo was carried out in a $80{\mu}l$ drop with $Ca^{2+}-Mg^{2+}$ free D-PBS and the viability of biopsied embryos were evaluated in IVMD (IFP, Japan) medium at 12 hrs culture time. The sex ratio of biopsied Hanwoo embryos were male vs. female of $43.5\%\;vs.\;56.5\%$ in vivo and $33.9\%\;vs.\;49.2\%$ in vitro respectively, and male rate of biopsied Holstein embryos were significantly higher than female $(70.8\;vs.\;29.2\%)$. and indefinite rate of in vitro embryos was $16.9\%$ and in vivo was not. The degeneration rate of biopsied embryo, in vitro embryos were significantly higher than in vivo $(13.2\%\;vs,\;0.0\%,\;p<0.05)$. The survivability of in vivo embryo were between biopsied following punching method was significantly (P<0.05) higher than bisection method produced embryos $(100\%\;vs.\;83.3\%)$ and in vitro had no difference. However, the degeneration rate of biopsied embryo by bisection method was significantly higher than punching methods between in vivo and in vitro $(16.7\;vs.\;22.6\%,\;respectively,\;p<0.05)$. In conclusion, these results indicate that punching method was optimal and survivability after embryo biopsy was useful for reducing the damage caused by the embryo biopsy procedure for LAMP-based embryo sexing.